We propose to develop and configure assays for high through put screening (HTS) for two acetyl transferases. The first, glucosamine-1-phosphate acetyl transferase (GAT) is an undeveloped target in peptidoglycan synthesis. It is essential for eubacterial growth, and its lack of function results in cell lysis. We propose to develop this assay using M. tuberculosis GAT enzyme (which is part of the bi-functional GlmU protein). Inhibitors of TB GAT are especially needed because there are no good peptidoglycan targeting antibiotics available for TB. Also new drugs against TB are needed because of the magnitude of the disease world wide and the emergence of drug resistant strains. We also propose to develop and configure assays for HTS of serine acetyl transferase (SAT) again using TB SAT. SAT is required for the synthesis of cysteine and has a role in bacterial extracellular signaling. It is not found in humans but is found in bacteria and several lower eukaryotic pathogens. Thus targeting SAT takes a novel approach to new drug development by hypothesizing that many pathogens, if unable to make cysteine, will not get adequate supplies from the host. Inhibitors of this enzyme, as can be found by HTS, are needed to validate this hypothesis. Both enzymes use acetyl-CoA to acetylate their substrate and the assay for both enzymes relies on the detection of free CoA produced by them. CoA is detected via its free SH group by forming a fluorescent adduct. We have preliminary data that this approach works for GAT and propose to develop it for GAT and extend it to SAT. After each assay is developed we propose to configure them for HTS by minimization of background, minimization of costs, demonstration of acceptable statistical parameters, and automation via a robotic liquid handler. We then propose to screen between 5 and 20 thousand compounds to test each assay. Future research will include submission of the assays to the NIH roadmap initiative for screening of large numbers of compounds to allow refinement of hits to candidates for preclinical development of new drugs. Relevance to Public Health: New drugs against bacteria and parasites are needed because drug resistance is resulting in current drugs not working. The work proposed herein will aid in the development of such new drugs by working with NIH to find chemical candidates that can be developed into new drugs.